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Measurement of hemoglobin in long-term fixed erythroid cells: application development for cell science experiments in microgravity

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Methods in Cell Science

Abstract

Studying the effects of microgravity on cell differentiation will enhance our understanding of fundamental biology and is indispensable for a sustained space program. Rauscher murine erythroleukemic cells were chosen as a model system to study erythroid cell differentiation aboard the International Space Station because these cells undergo differentiation in response to the natural inducer, erythropoietin, as well as various chemical inducers. We have now developed a method to quantify hemoglobin in Rauscher cells after weeks of fixation and storage required for such space biology experiments. By exploiting the pseudoperoxidase activity of hemoglobin and by using reagents that yield a soluble chromophore that freely passes out of fixed cells, we developed a highly specific and sensitive assay applicable to cells fixed as long as 4 months.

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References

  1. Watowich SS, Yoshimura A, Longmore GD, Hilton DJ, Yoshimura Y, Lodish HF (1992). Homodimerization and constitutive activation of the erythropoietin receptor. Proc Natl Acad Sci USA 89(6): 2140–2144.

    Google Scholar 

  2. Miura O, Nakamura N, Quelle FW, Witthuhn BA, Ihle JN, Aoki N (1994). Erythropoietin induces association of the JAK2 protein tyrosine kinase with the erythropoietin receptor in vivo. Blood 84(5): 1501–1507.

    Google Scholar 

  3. Fujitani Y, Hibi M, Fukada T, Takahashi-Tezuka M, Yoshida H, Yamaguchi T, Sugiyama K, Yamanaka Y, Nakajima K, Hirano T (1997). An alternative pathway for STAT activation that is mediated by the direct interaction between JAK and STAT. Oncogene 14(7): 751–761.

    Google Scholar 

  4. Sytkowski AJ (2004). Erythropoietin: blood, brain and beyond. Weinheim: WILEY-VCH

  5. Nathan DG, Sytkowski AJ (1983). Editorial retrospective. Erythropoietin and the regulation of erythropoiesis. N Engl J Med 308(9): 520–522

    Google Scholar 

  6. Tavassoli M (1982). Anemia of spaceflight. Blood 60(5): 1059–1067

    Google Scholar 

  7. Alfrey CP, Rice L, Udden MM, Driscoll TB (1997). Neocytolysis: physiological down-regulator of red-cell mass. Lancet 349(9062): 1389–1390

    Google Scholar 

  8. Robertson D, Krantz SB, Biaggioni I (1994). The anemia of microgravity and recumbency: role of sympathetic neural control of erythropoietin production. Acta Astronaut 33: 137–141

    Google Scholar 

  9. Robertson D, Convertino VA, Vernikos J (1994). The sympathetic nervous system and the physiologic consequences of spaceflight: a hypothesis. Am J Med Sci 308(2): 126–132

    Google Scholar 

  10. Leach CS, Johnson PC, Cintron NM (1988). The endocrine system in space flight. Acta Astronaut 17(2): 161–166

    Google Scholar 

  11. Hollander J, Gore M, Fiebig R, Mazzeo R, Ohishi S, Ohno H, Ji LL (1998). Spaceflight downregulates antioxidant defense systems in rat liver. Free Radic Biol Med 24(2): 385–390

    Google Scholar 

  12. Plett PA, Frankovitz SM, Abonour R, Orschell-Traycoff CM (2001). Proliferation of human hematopoietic bone marrow cells in simulated microgravity. In Vitro Cell Dev Biol Anim 37(2): 73–78

    Google Scholar 

  13. Davis TA, Wiesmann W, Kidwell W, Cannon T, Kerns L, Serke C, Delaplaine T, Pranger A, Lee KP (1996). Effect of spaceflight on human stem cell hematopoiesis: suppression of erythropoiesis and myelopoiesis. J Leukoc Biol 60(1): 69–76

    Google Scholar 

  14. Sytkowski AJ, Davis KL (2001). Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the NASA rotating wall vessel bioreactor. In Vitro Cell Dev Biol-Animal 37(2): 79–83

    Google Scholar 

  15. De Both NJ, Vermey M, van’t Hull E, Klootwijk-van-Dijke E, van Griensven LJ, Mol JN, Stoof TJ (1978). A new erythroid cell line induced by Rauscher murine leukaemia virus. Nature 272(5654): 626–628

    Google Scholar 

  16. Sytkowski AJ, Salvado AJ, Smith GM, McIntyre CJ, De Both NJ (1980). Erythroid differentiation of clonal Rauscher erythroleukemia cells in response to erythropoietin or dimethyl sulfoxide. Science 210(4465): 74–76

    Google Scholar 

  17. Showers MO, Moreau JF, Linnekin D, Druker B, D’Andrea AD (1992). Activation of the erythropoietin receptor by the Friend spleen focus-forming virus gp55 glycoprotein induces constitutive protein tyrosine phosphorylation. Blood 80(12): 3070–3078

    Google Scholar 

  18. Friend C, Scher W, Holland JG, Sato T (1971). Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc Natl Acad Sci USA 68(2): 378–382

    Google Scholar 

  19. Singer D, Cooper M, Maniatis GM, Marks PA, Rifkind RA (1974). Erythropoietic differentiation in colonies of cells transformed by Friend virus. Proc Natl Acad Sci USA 71(7): 2668–2670

    Google Scholar 

  20. Leder A, Leder P (1975). Butyric acid, a potent inducer of erythroid differentiation in cultured erythroleukemic cells. Cell 5(3): 319–322

    Google Scholar 

  21. Orkin SH, Harosi FI, Leder P (1975). Differentiation in erythroleukemic cells and their somatic hybrids. Proc Natl Acad Sci USA 72(1): 98–102

    Google Scholar 

  22. Ebert PS, Wars I, Buell DN (1976). Erythroid differentiation in cultured Friend leukemia cells treated with metabolic inhibitors. Cancer Res 36(5): 1809–1813

    Google Scholar 

  23. Reuben RC, Wife RL, Breslow R, Rifkind RA, Marks PA (1976). A new group of potent inducers of differentiation in murine erythroleukemia cells. Proc Natl Acad Sci U S A 73(3): 862–866

    Google Scholar 

  24. Andersson LC, Jokinen M, Gahmberg CG (1979). Induction of erythroid differentiation in the human leukaemia cell line K562. Nature 278(5702): 364–365

    Google Scholar 

  25. Wu H (1923). Studies on hemoglobin: an ultra-micromethod for the determination of hemoglobin as a peroxidase. Biochem J Japan 2: 189–205

    Google Scholar 

  26. Lijana RC, Williams MC (1979). Tetramethylbenzidine – a substitute for benzidine in hemoglobin analysis. J Lab Clin Med 94(2): 266–276

    Google Scholar 

  27. Ralph PH (1941). The histochemical demonstration of hemoglobin in blood cells and tissue smears. Stain Technology 16(3): 105–106

    Google Scholar 

  28. Kaplow LS (1965). Simplified myeloperoxidase stain using benzidine dihydrochloride. Blood 26(2): 215–219

    Google Scholar 

  29. Wanda PE, Lee LT, Howe C (1981). A spectrophotometric method for measuring hemoglobin in erythroleukemic cells (K562). J. Histochem Cytochem 29(12): 1442–1444

    Google Scholar 

  30. Kiernan JA (2000). Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microsc Today 001: 8–12

    Google Scholar 

  31. Holland VR, Saunders BC (1974). A safer substitute for benzidine in the detection of blood. Tetrahedeon 30: 3299–3302

    Google Scholar 

  32. Frey A, Meckelein B, Externest D, Schmidt MA (2000). A stable and highly sensitive 3,3′,5,5′-tetramethylbenzidine-based substrate reagent for enzymelinked immunosorbent assays. J Immunol Methods 233(1–2): 47–56

    Google Scholar 

  33. Gahmberg CG, Andersson LC (1981). K562 – a human leukemia cell line with erythroid features. Semin Hematol 18(1): 72–77

    Google Scholar 

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Authors and Affiliations

  1. Division of Hematology and Oncology, Laboratory for Cellular and Molecular Biology, Beth, Israel

    Kun Xu

  2. Deaconess Medical Center, Department of Medicine Boston, Harvard Medical School, Massachusetts, 02215, USA

    Kerry L. Davis & Arthur J. Sytkowski

  3. Laboratory for Cellular and Molecular Biology, Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA, 02215, USA

    Arthur J. Sytkowski

Authors
  1. Kun Xu
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  2. Kerry L. Davis
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  3. Arthur J. Sytkowski
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Correspondence to Arthur J. Sytkowski.

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Xu, K., Davis, K.L. & Sytkowski, A.J. Measurement of hemoglobin in long-term fixed erythroid cells: application development for cell science experiments in microgravity. Methods Cell Sci 25, 247–252 (2004). https://doi.org/10.1007/s11022-004-2381-6

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  • DOI: https://doi.org/10.1007/s11022-004-2381-6

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